Mast cell histamine and cytokine assays.

Mast cells are crucial to the development of chronic allergic inflammation and are likely to play a critical role in host defense. In this chapter methodology for histamine and cytokine assays is provided. Crosslinkage of IgE receptor I (Fc epsilonRI) on cord blood-derived mast cells by myeloma IgE and anti-human IgE is used to induce histamine release. Histamine levels were measured in the culture supernatants using an enzyme-linked immunosorbent assay. A human mast cell line (HMC-1), derived from a patient with mast cell leukemia, was activated with interleukin (IL)-1beta to study cytokine production and gene expression. Cytokine gene expression was evaluated by reverse transcriptase polymerase chain reaction and cytokine production was assayed in culture supernatants using an enzyme-linked immunosorbent assay kit.

Induction of p38- and gC1qR-dependent IL-8 expression in pulmonary fibroblasts by soluble hepatitis C core protein.

BACKGROUND: Recent studies suggest that HCV infection is associated with progressive declines in pulmonary function in patients with underlying pulmonary diseases such as asthma and chronic obstructive pulmonary disease. Few molecular studies have addressed the inflammatory aspects of HCV-associated pulmonary disease. Because IL-8 plays a fundamental role in reactive airway diseases, we examined IL-8 signaling in normal human lung fibroblasts (NHLF) in response to the HCV nucleocapsid core protein, a viral antigen shown to modulate intracellular signaling pathways involved in cell proliferation, apoptosis and inflammation. METHODS: NHLF were treated with HCV core protein and assayed for IL-8 expression, phosphorylation of the p38 MAPK pathway, and for the effect of p38 inhibition. RESULTS: Our studies demonstrate that soluble HCV core protein induces significant increases in both IL-8 mRNA and protein expression in a dose- and time-dependent manner. Treatment with HCV core led to phosphorylation of p38 MAPK, and expression of IL-8 was dependent upon p38 activation. Using TNFalpha as a co-stimulant, we observed additive increases in IL-8 expression. HCV core-mediated expression of IL-8 was inhibited by blocking gC1qR, a known receptor for soluble HCV core linked to MAPK signaling. CONCLUSION: These studies suggest that HCV core protein can lead to enhanced p38- and gC1qR-dependent IL-8 expression. Such a pro-inflammatory role may contribute to the progressive deterioration in pulmonary function recently recognized in individuals chronically infected with HCV.

Obstructive sleep apnea, inflammation, and cardiopulmonary disease.

Obstructive sleep apnea (OSA) occurs commonly in the U.S. population and is seen in both obese as well as non-obese individuals. OSA is a disease characterized by periodic upper airway collapse during sleep, which then results in either apnea, hypopnea, or both. The disorder leads to a variety of medical complications. Neuropsychiatric complications include daytime somnolence, cognitive dysfunction, and depression. Increased incidence of motor vehicle accidents has been documented in these patients and probably reflects disordered reflex mechanisms or excessive somnolence. More importantly, vascular disorders such as hypertension, stroke, congestive cardiac failure, arrhythmias, and atherosclerosis occur frequently in these patients. The lungs may be affected by pulmonary hypertension and worsening of asthma. Recent data from several laboratories demonstrate that obstructive sleep apnea is characterized by an inflammatory response. Cytokines are elaborated during the hypoxemic episodes leading to inflammatory responses as marked clinically by elevated C-reactive protein (CRP). As elevated CRP levels are considered markers of the acute phase response and characterize progression of vascular injury in coronary artery disease, it is likely that obstructive sleep apnea could lead to worsening of vasculopathy. Moreover, as inflammatory mechanisms regulate bronchial asthma, it is also likely that cytokines and superoxide radicals generated during hypoxemic episodes could exacerbate reactive airway disease. Patients with Cough, Obstructive sleep apnea, Rhinosinusitis, and Esophageal reflux clustered together can be categorized by the acronym, "CORE", syndrome. The purpose of this manuscript is to review the inflammatory responses that occur in patients with obstructive sleep apnea and relate them to the occurrence of cardiopulmonary disease.

MAPK-dependent regulation of IL-1- and beta-adrenoreceptor-induced inflammatory cytokine production from mast cells: implications for the stress response.

BACKGROUND: Catecholamines, such as epinephrine, are elaborated in stress responses, and mediate vasoconstriction to cause elevation in systemic vascular resistance and blood pressure. Our previous study has shown that IL-1 can induce mast cells to produce proinflammatory cytokines which are involved in atherogenesis. The aim of this study was to determine the effects of epinephrine on IL-1-induced proatherogenic cytokine production from mast cells. RESULTS: Two ml of HMC-1 (0.75 x 106 cells/ml) were cultured with epinephrine (1 x 10-5 M) in the presence or absence of IL-1 beta (10 ng/ml) for 24 hrs. HMC-1 cultured alone produced none to trace amounts of IL-6, IL-8, and IL-13. IL-1 beta significantly induced production of these cytokines in HMC-1, while epinephrine alone did not. However, IL-6, IL-8, and IL-13 production induced by IL-1 beta were significantly enhanced by addition of epinephrine. The enhancing effect appears to involve NF-kappa B and p38 MAPK pathways. Flow cytometry showed the presence of beta1 and beta2 adrenoreceptors on resting mast cells. The enhancing effect of proatherogenic cytokine production by epinephrine was down regulated by the beta1 and beta2 adrenoceptor antagonist, propranolol, but not by the beta1 adrenoceptor antagonist, atenolol, suggesting the effect involved beta2 adrenoceptors. The enhancing effect of epinephrine on proatherogenic cytokine production was also down regulated by the immunosuppressive drug, dexamethasone. CONCLUSIONS: These results not only confirm that an acute phase cytokine, IL-1 beta, regulates mast cell function, but also show that epinephrine up regulates the IL-1 beta induction of proatherogenic cytokines in mast cells. These data provide a novel role for epinephrine, a stress hormone, in inflammation and atherogenesis.

Molecular regulation of interleukin-13 and monocyte chemoattractant protein-1 expression in human mast cells by interleukin-1beta.

Mast cells play pivotal roles in immunoglobulin (Ig) E-mediated airway inflammation, expressing interleukin (IL)-13 and monocyte chemoattractant protein-1 (MCP-1), which in turn regulate IgE synthesis and/or inflammatory cell recruitment. The molecular effects of IL-1beta on cytokine expression by human mast cells (HMC) have not been studied well. In this report, we provide evidence that human umbilical cord blood-derived mast cells (CBDMC) and HMC-1 cells express the type 1 receptor for IL-1. We also demonstrate that IL-1beta and tumor necrosis factor-alpha are able to induce, individually or additively, dose-dependent expression of IL-13 and MCP-1 in these cells. The induction of IL-13 and MCP-1 gene expression by IL-1beta was accompanied by the activation of IL-1 receptor-associated kinase and translocation of the transcription factor, nuclear factor (NF) kappaB into the nucleus. Accordingly, Bay-11 7082, an inhibitor of NF-kappaB activation, inhibited IL-1beta-induced IL-13 and MCP-1 expression. IL-1beta also induced IL-13 promoter activity while enhancing the stability of IL-13 messenger RNA transcripts. Dexamethasone, a glucocorticoid, inhibited IL-1beta-induced nuclear translocation of NF-kappaB and also the secretion of IL-13 from mast cells. Our data suggest that IL-1beta can serve as a pivotal costimulus of inflammatory cytokine synthesis in human mast cells, and this may be partly mediated by IL-1 receptor-binding and subsequent signaling via nuclear translocation of NF-kappaB. Because IL-1beta is a ubiquitously expressed cytokine, these findings have important implications for non-IgE-mediated signaling in airway mast cells as well as for innate immunity and airway inflammatory responses, such as observed in extrinsic and intrinsic asthma.

Eosinophilia in a patient with cyclical vomiting: a case report.

BACKGROUND: Eosinophilic gastritis is related to eosinophilic gastroenteritis, varying only in regards to the extent of disease and small bowel involvement. Common symptoms reported are similar to our patient's including: abdominal pain, epigastric pain, anorexia, bloating, weight loss, diarrhea, ankle edema, dysphagia, melaena and postprandial nausea and vomiting. Microscopic features of eosinophilic infiltration usually occur in the lamina propria or submucosa with perivascular aggregates. The disease is likely mediated by eosinophils activated by various cytokines and chemokines. Therapy centers around the use of immunosuppressive agents and dietary therapy if food allergy is a factor. CASE PRESENTATION: The patient is a 31 year old Caucasian female with a past medical history significant for ulcerative colitis. She presented with recurrent bouts of vomiting, abdominal pain and chest discomfort of 11 months duration. The bouts of vomiting had been reoccurring every 7-10 days, with each episode lasting for 1-3 days. This was associated with extreme weakness and cachexia. Gastric biopsies revealed intense eosinophilic infiltration. The patient responded to glucocorticoids and azathioprine. The differential diagnosis and molecular pathogenesis of eosinophilic gastritis as well as the molecular effects of glucocorticoids in eosinophilic disorders are discussed. CONCLUSIONS: The patient responded to a combination of glucocorticosteroids and azathioprine with decreased eosinophilia and symptoms. It is likely that eosinophil-active cytokines such as interleukin-3 (IL-3), granulocyte macrophage colony stimulating factor (GM-CSF) and IL-5 play pivotal roles in this disease. Chemokines such as eotaxin may be involved in eosinophil recruitment. These mediators are downregulated or inhibited by the use of immunosuppressive medications.

The role of human mast cell-derived cytokines in eosinophil biology.

Eosinophil-mediated diseases, such as allergic asthma, eosinophilic fasciitis, and certain hypersensitivity pulmonary disorders, are characterized by eosinophil infiltration and tissue injury. Mast cells and T cells often colocalize to these areas. Recent data suggest that mast cells can contribute to eosinophil-mediated inflammatory responses. Activation of mast cells can occur by antigen and immunoglobulin E (IgE) via the high-affinity receptor (FcepsilonRI) for IgE. The liberation of proteases, leukotrienes, lipid mediators, and histamine can contribute to tissue inflammation and allow recruitment of eosinophils to tissue. In addition, the synthesis and expression of a plethora of cytokines and chemokines (such as granulocyte-macrophage colony-stimulating factor [GM-CSF], interleukin-1 [IL-1], IL-3, IL-5, tumor necrosis factor-alpha [TNF-alpha], and the chemokines IL-8, regulated upon activation normal T cell expressed and secreted [RANTES], monocyte chemotactic protein-1 [MCP-1], and eotaxin) by mast cells can influence eosinophil biology. Stem cell factor (SCF)-c-kit, cytokine-cytokine receptor, and chemokine-chemokine receptor (CCR3) interactions leading to nuclear factor kappaB (NF-kappaB), mitogen-activated protein kinase (MAPK) expression, and other signaling pathways can modulate eosinophil function. Eosinophil hematopoiesis, activation, survival, and elaboration of mediators can all be regulated thus by mast cells in tissue. Moreover, because eosinophils can secrete SCF, eosinophils can regulate mast cell function in a paracrine manner. This two-way interaction between eosinophils and mast cells can pave the way for chronic inflammatory responses in a variety of human diseases. This review summarizes this pivotal interaction between human mast cells and eosinophils.

Dermatology for the practicing allergist: Tinea pedis and its complications.

Tinea pedis is a chronic fungal infection of the feet, very often observed in patients who are immuno-suppressed or have diabetes mellitus. The practicing allergist may be called upon to treat this disease for various reasons. Sometimes tinea infection may be mistaken for atopic dermatitis or allergic eczema. In other patients, tinea pedis may complicate allergy and asthma and may contribute to refractory atopic disease. Patients with recurrent cellulitis may be referred to the allergist/immunologist for an immune evaluation and discovered to have tinea pedis as a predisposing factor. From a molecular standpoint, superficial fungal infections may induce a type2 T helper cell response (Th2) that can aggravate atopy. Th2 cytokines may induce eosinophil recruitment and immunoglobulin E (IgE) class switching by B cells, thereby leading to exacerbation of atopic conditions. Three groups of fungal pathogens, referred to as dermatophytes, have been shown to cause tinea pedis: Trychophyton sp, Epidermophyton sp, and Microsporum sp. The disease manifests as a pruritic, erythematous, scaly eruption on the foot and depending on its location, three variants have been described: interdigital type, moccasin type, and vesiculobullous type. Tinea pedis may be associated with recurrent cellulitis, as the fungal pathogens provide a portal for bacterial invasion of subcutaneous tissues. In some cases of refractory asthma, treatment of the associated tinea pedis infection may induce remission in airway disease. Very often, protracted topical and/or oral antifungal agents are required to treat this often frustrating and morbid disease. An evaluation for underlying immuno-suppression or diabetes may be indicated in patients with refractory disease.

Inhibition of GM-CSF production in fibroblast-monocyte coculture by prednisone and effects of rhGM-CSF on human lung fibroblasts.

Fibroblasts play a sentinel role in asthmatic disease. They are the main constituents of connective tissue and are increased in number in the asthmatic lung. They are also capable of secreting a diverse repertoire of cytokines and are able to be activated by pro-inflammatory cytokines and cell-cell contact. Previously we have reported that normal human lung fibroblasts (NHLF) can be activated by monocytes (U937) through cell-cell contact to produce GM-CSF. Here we show that GM-CSF production from NHLF activated by monocyte contact is inhibited by prednisone, a synthetic glucocorticoid used in the treatment of asthma. GM-CSF is an acidic glycoprotein that potentiates development of cells in the granulocyte and macrophage lineage and is secreted at sites of peripheral inflammation. The receptor for GM-CSF was found on NHLF by flow cytometry and was able to be up-regulated by interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha and recombinant human (rh) GM-CSF. To test autocrine effects of GM-CSF on fibroblasts, rh GM-CSF was used in proliferation studies and was found to decrease fibroblast proliferation. Prednisone was used to block NF-kappaB activation and GM-CSF gene expression as well. These data indicate mechanism of action and treatment for cell-cell contact mediated inflammation of infiltrating monocytes with fibroblasts as seen in asthma and other diseases like graft versus host disease.

Human lung fibroblasts express interleukin-6 in response to signaling after mast cell contact.

Asthma is a chronic inflammatory disease of the airways. Mast cell-derived cytokines may mediate both airway inflammation and remodeling. It has also been shown that fibroblasts can be the source of proinflammatory cytokines. In the human airways, mast cell-fibroblast interactions may have pivotal effects on modulating inflammation. To study this further, we cocultured normal human lung fibroblasts (NHLF) with a human mast cell line (HMC-1) and assayed for production of interleukin (IL)-6, an important proinflammatory cytokine. When cultured together, NHLF/HMC-1 contact induced IL-6 secretion. Separation of HMC-1 and NHLF cells by a porous membrane inhibited this induction. HMC-1-derived cellular membranes caused an increase in IL-6 production in NHLF. Activation of p38 MAPK was also seen in cocultures by Western blot, whereas IL-6 production in cocultures was significantly inhibited by the p38 inhibitor SB203580. IL-6 production in cocultures was minimally inhibited by a chemical inhibitor of nuclear factor-kappaB (Bay11), indicating that nuclear factor-kappaB may have a minimal role in signaling IL-6 production in mast cell/fibroblasts cocultures. Blockade of inter-cellular adhesion molecule-1, tumor necrosis factor-RI, and surface IL-1beta with neutralizing antibodies failed to significantly decrease IL-6 production in our coculture, indicating that other receptor-ligand associations may be responsible for this activation. These novel studies reveal the importance of cell-cell interactions in the complex milieu of airway inflammation.

GM-CSF induction in human lung fibroblasts by IL-1beta, TNF-alpha, and macrophage contact.

Fibroblast-derived cytokines may play crucial roles in airway inflammation. In this study, we analyzed expression of the inflammatory cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), a major eosinophilopoietin, by normal human lung fibroblast (NHLF) cells and its regulation by monokines and macrophage contact. NHLFs were stimulated with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) and were cocultured with the U937 myelomonocytic cell line. The expression of GM-CSF transcripts was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), and GM-CSF protein was detected by ELISA. Nuclear translocation of nuclear factor-kappaB (NF-kappaB), an important transcription factor for inflammatory gene expression, was assessed by electrophoretic mobility shift assay (EMSA). Both IL-1beta and TNF-alpha significantly enhanced the production of GM-CSF by NHLF. Coculturing of peripheral blood mononuclear cells (PBMC) with NHLF induced GM-CSF expression. This phenomenon was also seen on coculturing U937 cells or membranes derived from U937 with NHLF but was inhibited when the two types of cells were separated, suggesting a need for cell-cell contact. U937 membranes, as well as IL-1beta and TNF-alpha, induced nuclear translocation of NF-kappaB. These data support a prominent role for macrophage-fibroblast interactions in airway inflammation and fibrosis.